CN114144206B

CN114144206B - Tissue fibrosis inhibitor using biocompatible polymer

Info

Publication number: CN114144206B
Application number: CN202080055061.1A
Authority: CN
Inventors: 嵯峨秀树; 乾美奈子; 小仓训子
Original assignee: Chemo Sero Therapeutic Research Institute Kaketsuken
Current assignee: Chemo Sero Therapeutic Research Institute Kaketsuken
Priority date: 2019-08-01
Filing date: 2020-07-31
Publication date: 2023-03-24
Anticipated expiration: 2040-07-31
Also published as: US20240216577A1; KR102673609B1; TW202214312A; EP4008368A4; KR20220044520A; CA3144487C; WO2021020576A1; CN114144206A; BR112022001136A2; JP7472142B2; EP4008368A1; US20220265896A1; JPWO2021020576A1; CA3144487A1

Abstract

The problem to be solved by the present invention is to provide a fibrosis-inhibiting agent that solves the problem of inhibiting fibrosis of the surface of an organ or tissue, particularly the epicardial surface. In addition, by inhibiting fibrosis, the present invention prevents or reduces the subsequent occurrence of adhesions to avoid organ or tissue damage during re-surgery. A fibrosis-inhibiting agent for inhibiting fibrosis of a tissue by fixing a biocompatible polymer to the tissue where the inhibition of fibrosis is desired is provided.

Description

Tissue fibrosis inhibitor using biocompatible polymer

Technical Field

The present invention relates to an inhibitor of fibrosis of a tissue after a surgical operation. More particularly, the present invention relates to fibrosis-inhibiting agents characterized by the use of biocompatible polymers, which are also useful in inhibiting the occurrence of tissue adhesions.

Background

Fibrosis is a phenomenon in which connective tissue accumulates in tissues, and is a result of excessive production and accumulation of connective tissue such as extracellular matrix mainly composed of collagen in tissues. Fibrosis is observed in the heart, lung, liver, pancreas, kidney, etc. Among other things, fibrosis in the heart causes myocardial sclerosis and impairs cardiac function, although fibrosis prevents cardiac rupture of the heart after myocardial cell death. As cardiac dysfunction persists, enlargement of the heart occurs to compensate for the cardiac dysfunction. Therefore, it is important to control fibrosis. When physical stress such as autologous tissue and artificial objects or chemical reactions is applied to the surface of an organ such as the epicardium (heart surface) or tissue, the formation of fibers on the surface of the tissue increases, so that the tissue enlarges. In reoperation, particularly in reoperation of the heart, epicardial visibility is significantly deteriorated due to fibrosis, and the orientation of coronary arteries becomes unclear, so that there is a risk of causing damage to organs and blood vessels or tissues such as the heart. No fibrosis inhibitor has been found to meet the needs of the surgeon. Therefore, there is a need to produce a fibrosis-inhibiting agent that can inhibit fibrosis of the surface of an organ or tissue, particularly the epicardial surface.

In addition to fibrosis, another risk that may lead to organ or tissue damage is tissue adhesion after surgery. Postoperative tissue adhesions occur in a variety of fields, including cardiology as well as digestive surgery, orthopedic surgery, obstetrics and gynecology, ophthalmology, and the like. For example, an adhesion in the abdominal cavity after an abdominal operation is a physiological bioremediation reaction, and it is difficult to absolutely prevent the occurrence of the adhesion. Adhesions that occur after surgery have a certain frequency that causes adhesive ileus. It is believed that this frequency is somewhat proportional to the duration of the procedure and the amount of bleeding. In order to prevent or reduce the occurrence of adhesion, it is necessary to minimize separation/bleeding and the duration of the operation through careful surgical operations of surgeons, prevent intraoperative contamination and minimize residual foreign materials, use absorbable sutures, perform proper drainage, prevent infection, perform early activities after the operation, and the like. In emergency contaminated surgery, adequate intra-abdominal irrigation and posture changes are also required, and antibiotics are used after surgery. However, although these countermeasures are effective for preventing adhesive ileus to some extent, it is impossible to completely prevent the occurrence of ileus by these countermeasures (non-patent document 1).

According to recent studies, it is said that: mesothelial cells, which are membranous tissue covering the surface of body cavities such as the chest, pericardium and abdominal cavity, produce interleukin-6 at the site of surgical stress; the neutrophil granulocytes generate tumor necrosis factor TNF-alpha and transforming growth factor TGF-beta through the action of interleukin-6; and mesothelial cells are replaced by fibrosis by the action of such TGF- β to serve as a host of adhesion formation (non-patent document 2).

In the field of cardiology, post-operative epicardial-chest wall adhesions or pericardial-epicardial adhesions increase the risk of damaging the heart or great vessels during reoperation (non-patent document 3).

In cardiac surgery, autologous pericardium is used. However, when closure cannot be achieved using autologous pericardium alone, various types of pericardial substitutes are used. Examples of the pericardial substitute include bovine pericardium, silicone-coated polyester fabric, and Polytetrafluoroethylene (PTFE) sheet (non-patent document 4).

In order to avoid damage to the heart and great vessels during cardiac tamponade in the acute postoperative phase and in sternotomy in reoperation in the late postoperative phase, PTFE sheets with a thickness of 0.1mm are often used as pericardial substitutes in the area of pericardial defects in humans after open heart surgery. The outcome of the reoperation at a late post-long-term postoperative period is quite different from the outcome of the operation in the case where the heart is covered with autologous pericardium. The non-absorbable PTFE sheet, which has a structure that is non-invasive to biological tissues, can be easily removed during re-surgery. However, both the heart side and the sternum side of the PTFE sheet are covered with fibrous tissues to such an extent that the anatomical structure of each fibrous tissue cannot be confirmed. In particular, in the case of finding the burden caused by the residual lesion, thickening of the fibrous tissue clearly occurs. When a sheet is supplemented over a wide area of the heart surface in the initial operation, it is difficult to observe any diastolic failure even if a diagnosis of Constrictive Pericarditis (CP) is not given (non-patent document 5). Further, experimental data on this case are also publicly known (non-patent documents 6 to 8).

When PTFE sheets were transplanted as a pericardial substitute, evidence of inflammation induced by a foreign body reaction was strongly observed, with the inflammatory reaction becoming significant 4 weeks after transplantation and diminishing 12 weeks after transplantation. As a result of the transplantation, a very thick fibrous connective tissue membrane was formed on the epicardial side of the PTFE sheet. Such a PTFE sheet is useful because the sheet covers the heart and great vessels so as not to damage the heart and great vessels during the sternotomy when re-surgery becomes necessary. However, in the PTFF sheet, the problem is the influence of foreign body reaction in the PTFE sheet when re-surgery is unnecessary, particularly diastolic failure, including CP. When the PTFE sheet is removed, the area bounded by the sheet can be identified, and thus the PTFE sheet is generally considered to cause little inflammation of the surrounding tissue. However, in reality, the PTFE sheet is likely to cause an inflammatory reaction (non-patent document 5). Further, it has been reported that epicardial fibrosis caused by a PTFE sheet impairs the visibility of the heart (non-patent document 9).

The fibrosis of the epicardium is induced not only by the PTFE sheet but also by a pericardial defect condition or an autologous pericardial closure condition at the time of excision of the autologous pericardium. In experiments using a rabbit model, the occurrence of fibrosis in the case of expanded polytetrafluoroethylene (ePTFE) closure and the case of pericardial defect when an autologous pericardium is excised has been reported (non-patent document 10). In experiments using a canine model, the occurrence of fibrosis in the case of ePTFE closure and the case of autologous pericardial closure has been reported (non-patent document 11).

Meanwhile, a case where fibrin glue as a biological tissue adhesive is used for adhesion inhibition purposes has also been reported (non-patent documents 12 and 13). However, fibrin glue cannot completely inhibit tissue adhesion (patent documents 1 to 3), and thus has not yet solved a serious problem that surgeons are concerned about.

Acellular biological tissues and acellular organs (hereinafter collectively referred to as "acellular tissues") are matrices made by removing cellular components from biological tissues or organs of human bodies or animals of different species, and have received attention as cell scaffold materials or wound healing promoting materials that can be used in transplantation or regenerative medicine. A component other than cells in a biological tissue, namely extracellular matrix (ECM), is conserved among biological species in terms of its structure and composition, and it is clearly found that the component does not induce immune rejection among almost all biological species. In addition, it is also well known that ECM participates in differentiation of cells, and it is reported that cells cultured on ECM in a specific tissue differentiate into cells of the tissue. Based on these findings, the decellularized tissue constituting the ECM does not cause immune rejection after transplantation, and it is expected that cells passing through the recipient will be regenerated (non-patent document 14).

Documents of the prior art

Patent document

Patent document 1 JP-T-11-502431

Patent document 2 JP-A-2001-327592

Patent document 3 JP-T-2003-500170

Patent document 4 JP-4092397

Patent document 5 JP-A1-2008-111530

Patent document 6 JP-A-2009-50297

Non-patent document

Non-patent document 1, tsuneo Fukushima et al, surgical Therapy, 2006;94 (6):919-924

Non-patent document 2 Uyama N. et al, sci Rep.2019; 26;9 (1):17558

Non-patent document 3, dobell AR, et al, ann Thorac Surg, 1984;37:273-8

Non-patent document 4: ozeren M. Et al, cardiovasc. Surg. 2002;10 (5):489-493

Non-patent document 5, tetsuya Kitagawa, PEDIATRIC CARDIOLOGY and CARDIAC SURGERY, 2004;20 (6):632-633

Non-patent document 6, liermann A. Et al, helv Chir acta. 1992;58:515-519

Non-patent document 7 Muralidharan S. Et al, J Biomed Mater Res. 1991;25:1201-1209

Non-patent document 8, bunton RW. Et al, J Thorac Cardiovasc surg. 1990;100:99-107

Non-patent document 9, jukka T. et al, interactive cardiac and systemic delivery.2011; 12 (2):270-272

Non-patent document 10 Kaushal S. Et al, thorac Cardiovasc Surg. 2011;141:789-795

Non-patent document 11, naito Y. Et al, J Thorac Cardiovasc Surg. 2008;135:850-856

Non-patent document 12 Takamichi Sato et al, obstetrics and Gynecology, 1990;57 (12):2398-2404

Non-patent document 13 Kiyoshi Okuda et al, the World of Obstetrics and Gynecology, 1993;45 (9):759-764

Non-patent document 14, akio Kishida, organ Biology, 2018;25 (1):27-34

Non-patent document 15 Singleyn J.M. et al, biomaterials, 2009, 30, 5409-5416

Non-patent document 16 Singelyn J.M, et al, J.am. Coll. Cardiol., 2012, 59, 751-763

Non-patent document 17 Sonya B. Et al, sci. Transl. Med., 2013, 5, 173ra25

Non-patent document 18 Sasaki S. et al, mol. Vis., 2009, 15, 2022-2028

Nonpatent document 19 Yoshihide H.et al, biomaterials, 2010, 31, 3941-3949

Non-patent document 20 Seiichi F. Et al, biomaterials, 2010, 31, 3590-3595

Non-patent document 21 Negishi J. Et al, J. Artif. Organs, 2011, 14, 223-231

Non-patent document 22 Hirashima M. Et al, J. Biochem., 2016, 159, 261-270

Non-patent document 23 Meta A. Et al, J. Biosci. Bioeng, 2015, 120, 432-437.

Summary of The Invention

Problems to be solved by the invention

The problems to be solved by the present invention are (1) to inhibit fibrosis of the surface of an organ or tissue, particularly (2) to inhibit fibrosis of the epicardial surface, and to provide a fibrosis-inhibiting agent that can solve these problems. Another problem is preventing and reducing the occurrence of subsequent adhesions by inhibiting fibrosis to avoid organ or tissue damage during re-surgery.

Solution to the problem

The present inventors have conducted extensive and intensive studies. As a result, the present inventors have found a fibrosis inhibitor by which a biocompatible polymer is fixed to a tissue to be inhibited from fibrosis of the tissue, thereby inhibiting fibrosis of the tissue, and they have found that the fibrosis inhibitor can solve these problems.

The present invention includes the following aspects:

(1) An inhibitor of tissue fibrosis comprising a biocompatible polymer.

(2) The fibrosis inhibitor according to (1), wherein the biocompatible polymer is fibrin or dextrin gel.

(3) A tissue fibrosis inhibition kit comprising a combination of a biocompatible polymer and a cell scaffold material.

(4) The fibrosis suppressing kit according to (3), wherein the biocompatible polymer is fixed to one tissue to be suppressed in tissue fibrosis, and a tissue defect site in the other tissue is repaired with the cytoskeletal material (prosthesis).

(5) The fibrosis inhibition kit according to (3) or (4), wherein the biocompatible polymer is fibrin or dextrin gel.

(6) The fibrosis inhibition kit according to any one of (3) to (5), wherein the cell scaffold material is a decellularized tissue.

(7) The fibrosis suppressing kit according to (6), wherein the decellularized tissue is a tissue prepared by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura, peritoneum, omentum majus, diaphragm, fascia, dermis and skin.

(8) The fibrosis inhibition kit according to any one of (3) to (7), which promotes self-organization of the cell scaffold material.

(9) The fibrosis suppressing kit according to any one of (3) to (8), wherein the tissue to be suppressed in tissue fibrosis is epicardium.

(10) A tissue fibrosis inhibition kit comprising a combination of a biocompatible polymer and a pericardial substitute.

(11) The fibrosis suppressing kit according to (10), wherein the biocompatible polymer is fixed to one tissue to be suppressed in tissue fibrosis, and a tissue defect site in the other tissue is repaired with the pericardium substitute.

(12) The fibrosis inhibition kit according to (10) or (11), wherein the biocompatible polymer is fibrin or dextrin gel.

(13) The fibrosis suppression kit according to any one of (10) to (12), wherein the pericardial substitute is a decellularized tissue.

(14) The fibrosis inhibition kit according to (13), wherein the decellularized tissue is a tissue made by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura mater, peritoneum, omentum majus, diaphragm, fascia, dermis, and skin.

(15) The fibrosis suppression kit according to any one of (10) to (14), which promotes self-organization of the pericardial substitute.

(16) The fibrosis suppressing kit according to any one of (10) to (15), wherein the tissue to be suppressed in fibrosis of the tissue is pericardium.

(17) An adhesion barrier kit (adhesion barrier kit) in which a biocompatible polymer is fixed to one tissue to be inhibited from tissue adhesion, and a tissue defect site in the other tissue is repaired with a cytoskeletal material or a pericardial substitute.

(18) The adhesion barrier kit of (17), wherein the biocompatible polymer is fibrin or dextrin gel.

(19) The adhesion barrier kit of (17), wherein the cell scaffold material or the pericardial substitute is decellularized tissue.

(20) The adhesion barrier kit according to (19), wherein the decellularized tissue is a tissue prepared by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura, peritoneum, omentum majus, diaphragm, fascia, dermis, and skin.

(21) The adhesion barrier kit of any one of (17) to (20), which facilitates self-organization of the cell scaffold material or the pericardial substitute.

(22) The adhesion barrier kit according to any one of (17) to (21), wherein the tissue to be inhibited from adhesion is epicardium.

Effects of the invention

The fibrosis inhibitor according to the present invention has an effect of inhibiting fibrosis of the surface of an organ or tissue. The fibrosis inhibitor of the present invention inhibits fibrosis of the surface of an organ or tissue, particularly, the epicardial surface, to improve the epicardial visibility, and thus can reduce damage to the heart or blood vessels during the reoperation. In addition, the fibrosis inhibitor of the present invention can inhibit inflammatory reactions that induce fibrosis. Therefore, the fibrosis inhibitor is also expected to have an effect of reducing adhesions associated with inflammatory reactions and to be useful as an adhesion inhibitor.

Brief Description of Drawings

Fig. 1 is a photograph showing evaluation of rabbit cardiac fibrosis model (fibrosis inhibitor group) after observation for 3 months.

Fig. 2 is a photograph showing an evaluation of rabbit cardiac fibrosis model (fibrosis-inhibiting kit group) after 3 months of observation.

Fig. 3 is a photograph showing the evaluation of rabbit cardiac fibrosis model (control/pericardial defect) after 3 months of observation.

Fig. 4 is a photograph showing the evaluation of a rabbit cardiac fibrosis model (control/autologous pericardial closure) after 3 months of observation.

Fig. 5 is a photograph showing evaluation of rabbit cardiac fibrosis model (acellular tissue alone group) after 3 months of observation.

Fig. 6 shows an example of a method using the fibrosis inhibition kit and the adhesion barrier kit (a case in which (1) fibrin is applied and fixed to the epicardium of the heart, and then (2) decellularized tissue is sutured and fixed to a pericardial defect site located between the epicardium and the sternum).

Fig. 7 is a photograph showing the pathology of a rabbit heart model (fibrosis inhibition kit and adhesion barrier kit set) after 3 months of observation.

Fig. 8 is a photograph showing the pathology of a rabbit heart model (acellular tissue alone group) after 3 months of observation.

Fig. 9 is a photograph showing the pathology of a rabbit heart model (fibrin- (decellularized tissue) complex) after 3 months of observation.

Embodiments of the invention

The invention comprises the following steps: biocompatible polymers useful as fibrosis inhibitors; and a cell scaffold material.

The term "biocompatible polymer" as used herein refers to a polymer that produces a lesser immune response in vivo. Examples of biocompatible polymers include, but are not limited to, alginic acid, chitin, chitosan, glycosaminoglycans, collagen, chondroitin sulfate, cellulose, gelatin, dextran, dextrin, glycoproteins, hyaluronic acid, fibrinogen, fibrin, fibronectin, heparin, and heparinoids. Fibrin or dextrin gels are particularly preferred as biocompatible polymers.

The term "cell scaffold material" as used herein refers to a material that promotes cell adhesion/proliferation.

The decellularized tissue can be obtained by collecting a biological tissue from an animal (donor) and then subjecting the biological tissue to a decellularization treatment. Cells or pathogens (viruses, bacteria) from a donor can be removed from biological tissue by subjecting the biological tissue to a decellularization process. Thus, the occurrence of a heterologous immune response can be suppressed even when the biological tissue is transplanted into an animal of a species different from the donor species. For these reasons, there is no particular limitation on the type of animal used for collecting the biological tissue. At the same time, biological tissue is preferably easily obtained. Accordingly, the animal is preferably a non-human animal, and particularly preferably a mammalian livestock or an avian livestock. Examples of mammalian livestock include cattle, horses, camels, llamas, donkeys, yaks, sheep, pigs, goats, deer, alpacas, dogs, racoon dogs, weasels, foxes, cats, rabbits, hamsters, guinea pigs, rats, squirrels, and racoons. Examples of avian domestic animals include parakeets, parrots, chickens, ducks, turkeys, geese, guinea fowl, pheasants, ostriches and quails. Among these animals, pigs are preferred from the viewpoint of availability.

An example of a biological tissue is a tissue that has an extracellular matrix structure and can be subjected to a decellularization process. Examples of tissues are tissues selected from small intestine submucosa, bladder, amnion, dura, peritoneum, diaphragm, fascia, dermis, skin, liver, kidney, ureter (urinary duct), urethra, tongue, tonsil, esophagus, stomach, omentum major, small intestine, large intestine, anus, pancreas, heart, pericardium, blood vessel, spleen, lung, brain, bone, spinal cord, cartilage, testis, uterus, fallopian tube, ovary, placenta, cornea, skeletal muscle, tendon, nerve, and the like. When the decellularized tissue has a sheet form, the decellularized tissue can be easily applied to a living body. Thus, a preferred example of such a tissue is a tissue selected from the group consisting of small intestine submucosa, bladder, amniotic membrane, dura mater, peritoneum, greater omentum, diaphragm, fascia, dermis, and skin. Acellular tissue that does not have a sheet form is not unacceptable because the acellular tissue only has to be processed into a sheet form.

As a method of collecting biological tissue from an animal, a method suitable for an animal species or biological tissue of a donor can be used. Biological tissue collected from an animal is subjected to a decellularization process. The decellularization treatment is not particularly limited as long as cells or pathogens derived from a donor can be removed. Examples of the method include treatment with a surfactant (non-patent documents 15 to 21, patent documents 4 to 6), and the method can be appropriately selected depending on the animal species of the donor and the type of biological tissue.

In combining the biocompatible polymer with the decellularized tissue, the fixation of the biocompatible polymer to the tissue in which the tissue fibrosis is to be inhibited can be achieved by gelation of the biocompatible polymer itself. Examples of the method for immobilizing fibrin, which is one of biocompatible polymers, include: a method in which fibrin is fixed to a tissue to be inhibited from fibrosis by applying the fibrin to the tissue; and a method in which fibrin is fixed to a tissue by spraying powder (fibrinogen powder, thrombin powder) as a fibrin constituent onto the tissue. For example, when pericardium is defective in cardiac surgery, fibrosis of the epicardium or the like becomes a problem. To inhibit epicardial fibrosis, an example is the fixation of fibrin to the epicardium, while the decellularized tissue is sutured and fixed to the autologous pericardium that is left to reduce contact with the sternum. Fibrin is fixed to a tissue to be inhibited from fibrosis, more specifically, epicardium, while repairing a pericardial defect site located between the epicardium and the sternum with decellularized tissue. In this way, it can function as a fibrosis-inhibiting kit. In the fixation of fibrin, it is possible to apply either fibrinogen or thrombin first, followed by the other, or both simultaneously. Spray application using a spray device is also optional. The fixation of fibrin is completed when a slight turbidity of the transparent gel is observed. As described above, an example of a tissue in which tissue fibrosis is to be inhibited is the epicardium. However, examples of tissue are not limited to epicardium. In addition to the epicardium, it is believed that the occurrence of fibrosis can be suppressed in tissues of the lung and the like.

The concentration of fibrinogen or thrombin at the time of applying fibrin is not particularly limited as long as fibrin can be formed. For example, the concentration of fibrinogen is 4 mg/mL to 160 mg/mL, preferably 10 mg/mL to 80 mg/mL. For example, the concentration of thrombin is 1U/mL to 1200U/mL, preferably 60U/mL to 600U/mL.

Fibrin is a gel-like coagulation formed by the interaction of fibrinogen and thrombin as an enzyme, and is a pharmaceutical product that can be used for tissue closure, adhesion of damaged sites in organs, hemostasis, and the like. The type of fibrin is not particularly limited, and may be fibrin in which constituent components such as fibrinogen and thrombin are derived from blood, or may be prepared by recombinant techniques. One preferred example is BOLHEAL (registered trademark, KM Biologics Co., ltd.) for tissue adhesion.

The invention also includes a fibrosis-inhibiting kit in which fibrin and decellularized tissue are separately prepared. The kit is configured for application of fibrin and decellularized tissue by a physician at the time of use. When the decellularized tissue is lyophilized, the decellularized tissue is impregnated with a solvent to reconstitute the decellularized tissue and the reconstituted decellularized tissue is applied to the affected site. Thus, the kit may include a solvent for reconstitution.

The animal used for making the cardiac fibrosis model is not particularly limited, and is preferably a rabbit, more preferably a Japanese white rabbit of 5 to 6 months old. In the fabrication of the model, the thorax of the animal was opened under anesthesia through an incision, and then a metal dissector (raspatory) was brought into contact with the heart or epicardium. The duration of contact with the metal stripper is not particularly limited, and is, for example, 10 to 20 minutes. After treatment with a metal stripper, the heart can be dried with a desiccator (by supplying air).

One example of a method of assessing fibrosis inhibition is by visual inspection of the rating. For example, in the case of evaluating fibrosis inhibition in the epicardium, the evaluation can be made by rating a fibrosis grade of 0 (no fibrosis observed), a fibrosis grade of 1 (pericardium can be visually observed), and a fibrosis grade of 2 (epicardium cannot be visually observed). In this evaluation method, the determination may be made based on whether or not the blood vessels on the epicardium can be visually observed. When the epicardium is whitened and no blood vessel is observed, it can be determined that fibrosis is not satisfactorily inhibited. Further, a method in which the region of fibrosis grade 2 is compared with a control group to evaluate the fibrosis-inhibiting effect may also be preferably used.

The phrase "self-organization of a cell scaffold material or a pericardial substitute" as used herein means that an artificial material or the like is reconstituted into a tissue such as an autologous tissue after the artificial material or the like is transplanted into a living body, with a consequent reduction in immune response.

Examples

The present invention is described in more detail below by way of examples, but is not intended to limit the scope of the present invention in any way.

Example 1

Fibrosis-inhibiting effects of fibrosis-inhibiting agent and fibrosis-inhibiting kit:

as the decellularized tissue, decellularized porcine small intestine submucosa, decellularized fetal rabbit skin, and decellularized rabbit pericardium were used.

As biocompatible polymer, fibrin is used. Regarding blood-derived fibrin, a fibrinogen solution was prepared in BOLHEAL (registered trademark, KM Biologics Co., ltd.) for tissue adhesion by using fibrinogen freeze-dried powder and fibrinogen solubilization. A thrombin solution was prepared in BOLHEAL (registered trademark, KM Biologics co., ltd.) for tissue adhesion by using thrombin lyophilized powder and thrombin dissolution (dissolution). Recombinant fibrin was produced using recombinant fibrinogen (non-patent document 22) and recombinant thrombin (non-patent document 23), both produced by KM Biologics co. As the dextrin gel, adSpray (registered trademark, terumo Corporation) was used. When the transparent gel was slightly turbid, it was confirmed that the immobilization of fibrin was completed, and when a white gel containing microbubbles was formed, it was confirmed that the immobilization of dextrin was completed.

A cardiac fibrosis model was made using male japanese white rabbits of 5 to 6 months of age. An endotracheal tube was inserted into the trachea under anesthesia with xylazine and ketamine hydrochloride, and then connected to an artificial respirator. A portion (5 cm) of the sternum is cut around the notch in the second to fifth costal cartilage. The pericardium (2.0 cm x 1.5 cm) immediately below the incised thoracic portion was incised. The epicardium was contacted with a metal stripper for 10 minutes. The sternum, muscle and skin in the incised thoracic wound were sutured and closed. The appropriate timing of assessment is considered to be the time after inflammation in the tissue has ended and fibrosis has completed. Then, long-term evaluation was performed in such a manner that the animals were kept for 3 months in a conventional manner, then the thoracic cavity was incised and the grade of fibrosis of the epicardium was visually evaluated.

Fibrosis was rated according to the following criteria:

fibrosis rating 0 no fibrosis was observed.

Fibrosis rating 1 epicardium can be visually observed.

Fibrosis rating 2 epicardium was not visually observed.

The fibrosis-inhibiting effect can be determined by comparing the fibrosis grade 2 region with a control group. The region (%) of fibrosis level 2 was calculated according to the following formula:

(area of fibrosis grade 2)/(area of pericardial defect site) × 100.

In example 1, the following test groups were compared and evaluated.

(1) Fibrosis inhibitor group (blood-derived fibrin)

(2) Fibrosis-inhibiting reagent kit (blood-derived fibrin and acellular porcine small intestine submucosa)

(3) Fibrosis-inhibiting reagent kit (recombinant fibrin and acellular porcine small intestine submucosa)

(4) Fibrosis-inhibiting reagent kit (recombinant fibrin and acellular fetal rabbit skin)

(5) Fibrosis inhibiting reagent kit (recombinant fibrin and acellular rabbit heart pack)

(6) Fibrosis-inhibiting reagent kit (dextrin gel and decellularized porcine small intestine submucosa)

(7) Control group (pericardial defect)

(8) Control group (autologous pericardial closure)

(9) Acellular tissue alone (acellular porcine small intestine submucosa)

(10) Fibrin-acellular tissue complex (blood-derived fibrin complexed with decellularized porcine small intestine submucosa).

Groups (1) to (6) are groups of the present invention, and groups (7) to (10) are control groups. With respect to the fibrin application method used in (1) to (5), a fibrinogen solution and a thrombin solution are mixed together and the resulting mixture is applied to the epicardium of the pericardial defect site using an application device, wherein the final concentrations of the fibrinogen solution and the thrombin solution are 40 mg/mL and 125U/mL, respectively, and the applied solutions are fixed. The dextrin gel preparation method and dextrin gel application method for (6) were performed as specified in the package insert of AdSpray. With respect to the acellular tissue application method for (2) to (6) and (9), the acellular tissue is sutured and fixed (repaired) to the remaining autologous pericardium in such a manner that the acellular tissue is fixed to the pericardial defect site located between the epicardium and the sternum. With respect to the application sequence for (2) to (6), fibrin or dextrin gel was fixed to the epicardium, followed by suturing and fixing (repair) of the decellularized tissue to the autologous pericardium. Groups (7) to (10) are groups in each of which no fibrosis inhibitor was applied. Group (10) is a group in which fibrin is complexed with decellularized tissue. The fibrinogen solution and the thrombin solution are mixed together and the resulting mixture is applied to both sides of the decellularized tissue to complex these components with each other, and the resulting product is sutured and fixed (repaired) to the remaining autologous pericardium in such a manner that the resulting product is fixed to the pericardial defect site located between the epicardium and the sternum. The evaluation results are shown in table 1.

As is apparent from the above results, when the fibrosis-inhibiting agent and the fibrosis-inhibiting kit, which are the products of the present invention, are used, an excellent fibrosis-inhibiting effect is confirmed. In each of the fibrosis inhibitors of the present invention, when (3) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized porcine small intestine submucosa) was compared with (7) the control group (pericardial defect), a significant difference was confirmed by Wilcoxon rank-sum test (p < 0.01). Significant differences (p < 0.05) were confirmed when (1) the fibrosis inhibitor group (blood-derived fibrin), (2) the fibrosis-inhibiting reagent kit group (blood-derived fibrin and decellularized porcine small intestine submucosa), (4) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized rabbit skin), and (5) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized rabbit pericardium) were each compared to (7) the control group (pericardial defect). Significant differences were confirmed when (3) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized porcine small intestine submucosa) was compared to (8) the control group (autologous pericardial closure) (p < 0.05). When (2) the fibrosis-inhibiting reagent kit group (blood-derived fibrin and decellularized porcine small intestine submucosa), (3) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized porcine small intestine submucosa), (4) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized rabbit skin), and (5) the fibrosis-inhibiting reagent kit group (recombinant fibrin and decellularized rabbit pericardium) were each compared with (10) the fibrin-decellularized tissue complex group (blood-derived fibrin and decellularized porcine small intestine submucosa complex), a significant difference (p < 0.01) was confirmed. Significant differences were confirmed (p < 0.05) when comparing (1) the fibrosis inhibitor group (blood-derived fibrin) and (6) the fibrosis inhibitor reagent kit group (dextrin gel and decellularized porcine small intestine submucosa) with (10) the fibrin-decellularized tissue complex group (blood-derived fibrin complexed with decellularized porcine small intestine submucosa).

Example 2

Adhesion inhibition effect of adhesion barrier kit:

As biocompatible polymer, fibrin is used. Regarding blood-derived fibrin, a fibrinogen solution was prepared in BOLHEAL (registered trademark, KM Biologics Co., ltd.) for tissue adhesion by using fibrinogen freeze-dried powder and fibrinogen solubilization. A thrombin solution was prepared in BOLHEAL (registered trademark, KM Biologics co., ltd.) for tissue adhesion by using thrombin lyophilized powder and thrombin dissolution (dissolution). Recombinant fibrin was produced using recombinant fibrinogen (non-patent document 22) and recombinant thrombin (non-patent document 23), both produced by KM Biologics co. As the dextrin gel, adSpray (registered trademark, terumo Corporation) was used. When the transparent gel was slightly turbid, fixation of fibrin was confirmed to be completed, and when a white gel containing microbubbles was formed, fixation of dextrin was confirmed to be completed.

A heart tissue adhesion model was made using 5 to 6 month-old male japanese white rabbits. An endotracheal tube was inserted into the trachea under anesthesia with xylazine and ketamine hydrochloride, and then connected to an artificial ventilator. A portion (5 cm) of the sternum is cut around the notch in the second to fifth costal cartilage. The pericardium (2.0 cm x 1.5 cm) immediately below the incised thoracic portion was incised. The epicardium was contacted with a metal stripper for 10 minutes. The sternum, ribs, muscles and skin in the incised thoracic wound were sutured and closed. The appropriate timing of assessment is considered to be the time after inflammation in the tissue has ended and the adhesion is complete. Then, long-term evaluation was performed in such a manner that animals were kept for 3 months in a conventional manner, then the thoracic cavity was incised and the adhesion of epicardial injury sites (injured sites) was evaluated.

Adhesions were rated according to the following criteria, depending on the degree of separation of adhesions at the site of epicardial injury.

Adhesion rating 0: no adhesion

Adhesion rating 1 mild adhesion (adhesion does not require blunt dissection to separate the space between the reconstructed pericardium and sternum or epicardium)

Adhesion rating 2 moderate adhesion (adhesion requires blunt dissection to separate the space between the reconstructed pericardium and sternum or epicardium)

Adhesion grade 3 severe adhesion (adhesion requires sharp separation to separate the space between the reconstructed pericardium and sternum or epicardium).

The adhesion-inhibiting effect was determined by comparing the epicardial region of adhesion grade 0 and the epicardial region of adhesion grade 3 with the control group. The epicardial region (%) of adhesion grade 0 was calculated according to the following formula:

(epicardial area of adhesion grade 0)/(area of pericardial defect site) × 100.

The epicardial region (%) of adhesion grade 3 was calculated according to the following formula: (epicardial area of adhesion grade 3)/(area of pericardial defect site) × 100.

In example 2, the following test groups were compared and evaluated.

(1) Adhesion barrier reagent kit (blood-borne fibrin and acellular submucosa of small intestine of pig)

(2) Adhesion barrier reagent kit (recombinant fibrin and acellular porcine small intestine submucosa)

(3) Adhesion barrier reagent kit (recombinant fibrin and acellular fetal rabbit skin)

(4) Adhesion barrier reagent kit (recombinant fibrin and acellular rabbit heart pack)

(5) Adhesion barrier reagent kit (dextrin gel and acellular porcine small intestine submucosa)

(6) Control group (pericardial defect)

(7) Control group (autologous pericardial closure)

(8) Fibrin-acellular tissue complex (blood-derived fibrin is complexed with acellular porcine small intestine submucosa).

Groups (1) to (5) are groups of the present invention, and groups (6) to (8) are control groups. With respect to the fibrin application method for (1) to (4), a fibrinogen solution and a thrombin solution are mixed together and the resulting mixture is applied and fixed to the epicardium of the pericardial defect site using an application device, wherein the final concentrations of the fibrinogen solution and the thrombin solution are 40 mg/mL and 125U/mL, respectively. The dextrin gel preparation method and application method for (5) were carried out as specified in the package insert of AdSpray. With respect to the acellular tissue application method for (1) to (5), the acellular tissue is sutured and fixed (repaired) to the remaining autologous pericardium to be fixed to the pericardial defect site located between the epicardium and the sternum. With respect to the application sequence for (1) to (5), fibrin or dextrin gel was fixed to the epicardium, followed by suturing and fixing of the decellularized tissue to the autologous pericardium. Groups (6) to (8) are groups in each of which the adhesion barrier kit was not applied. Group (8) is a group in which fibrin is complexed with decellularized tissue. The fibrinogen solution and the thrombin solution are mixed together and the resulting mixture is applied to both sides of the decellularized tissue to complex these components with each other, and the resulting product is sutured and fixed (repaired) to the remaining autologous pericardium in such a manner that the resulting product is fixed to the pericardial defect site located between the epicardium and the sternum. The evaluation results are shown in table 2.

As is apparent from the above results, when the adhesion barrier kit as a product of the present invention was used, an excellent adhesion-inhibiting effect was confirmed over a long period of time. The region (%) of adhesion grade 0 in the epicardium was compared. As a result, when compared between (3) the adhesion barrier kit group (recombinant fibrin and decellularized rabbit skin) and (6) the control group (pericardial defect), significant differences in the adhesion barrier kit of the present invention were confirmed by Wilcoxon rank-sum test (p < 0.01). Significant differences were confirmed when (1) the adhesion barrier kit set (blood-derived fibrin and decellularized porcine small intestine submucosa) and (2) the adhesion barrier kit set (recombinant fibrin and decellularized porcine small intestine submucosa) were each compared to (6) the control group (pericardial defect) (p < 0.05). Significant differences were confirmed (p < 0.05) when comparing (3) the adhesion barrier kit group (recombinant fibrin and decellularized rabbit skin) with (7) the control group (autologous pericardial closure). Significant differences were confirmed when (1) the adhesion barrier kit group (blood-derived fibrin and decellularized porcine small intestine submucosa) and (2) the adhesion barrier kit group (recombinant fibrin and decellularized porcine small intestine submucosa) were each compared to (8) the fibrin-decellularized tissue complex group (blood-derived fibrin complexed with decellularized porcine small intestine submucosa) (p < 0.05). The region (%) of adhesion grade 3 in the epicardium was compared. As a result, when (1) the adhesion barrier kit group (blood-derived fibrin and decellularized porcine small intestine submucosa), (2) the adhesion barrier kit group (recombinant fibrin and decellularized porcine small intestine submucosa), (3) the adhesion barrier kit group (recombinant fibrin and decellularized fetal rabbit skin), (4) the adhesion barrier kit group (recombinant fibrin and decellularized rabbit epicardium), and (5) the adhesion barrier kit group (dextrin gel and decellularized porcine small intestine submucosa) were each compared with (6) the control group (pericardial defect), a significant difference (p < 0.01) was confirmed in the adhesion barrier kit of the present invention by Wilcoxon rank and test. Significant differences were confirmed (p < 0.05) when comparing (2) the adhesion barrier kit group (recombinant fibrin and decellularized porcine small intestine submucosa) to (7) the control group (autologous pericardial closure).

Example 3

Promotion of self-organization of decellularized tissue by fibrosis inhibition kit and adhesion barrier kit:

as the decellularized tissue, decellularized porcine small intestine submucosa was used.

As biocompatible polymer, fibrin is used. Regarding blood-derived fibrin, a fibrinogen solution was prepared in BOLHEAL (registered trademark, KM Biologics Co., ltd.) for tissue adhesion by using fibrinogen freeze-dried powder and fibrinogen solubilization. A thrombin solution was prepared in BOLHEAL (registered trademark, KM Biologics co., ltd.) for tissue adhesion by using thrombin lyophilized powder and thrombin dissolution (dissolution). Recombinant fibrin was produced using recombinant fibrinogen (non-patent document 22) and recombinant thrombin (non-patent document 23), both produced by a genetic engineering technique from KM Biologics co. When the transparent gel was slightly turbid, fixation of fibrin was confirmed to be completed, and when a white gel containing microbubbles was formed, fixation of dextrin was confirmed to be completed.

The acellular tissue was evaluated for self-organization using a rabbit heart model. Male japanese white rabbits 5 to 6 months old were used. An endotracheal tube was inserted into the trachea under anesthesia with xylazine and ketamine hydrochloride, and then connected to an artificial respirator. A portion (5 cm) of the sternum is cut around the notch in the second to fifth costal cartilage. The pericardium (2.0 cm. Times.1.5 cm) just below the part of the thorax that was incised. The epicardium was contacted with a metal stripper for 10 minutes. The sternum, ribs, muscles and skin in the incised thoracic wound were sutured and closed. For long-term evaluation, rabbits were routinely raised for 3 months and then the chest was dissected to collect acellular tissue. To assess the strength of the decellularized tissue, the rupture of the decellularized tissue at the time of collection was assessed. The incidence of rupture (%) was calculated according to the following formula:

(number of individuals in which rupture of decellularized tissue occurred at the time of collection)/(number of individuals in each test group). Times.100.

Histopathological specimens of the collected decellularized tissues were prepared and then HE stained. The degree of self-organization was evaluated in this manner. The pathology pictures of the results are shown in fig. 7 to 9.

In example 3, the following test groups were evaluated.

(1) Fibrosis inhibiting and adhesion barrier kit (blood-borne fibrin and decellularized porcine small intestine submucosa)

(2) Acellular tissue alone (acellular porcine small intestine submucosa)

(3) Fibrin-acellular tissue complex (blood-derived fibrin complexed with decellularized porcine small intestine submucosa).

With respect to the method of applying the decellularized tissue in (1) and (2), the decellularized tissue is sutured and fixed (repaired) to the remaining autologous pericardium to fix the decellularized tissue to the pericardial defect site located between the epicardium and the sternum. Group (3) is a group in which fibrin is complexed with decellularized tissue. The composite product is sutured and fixed (repaired) to the remaining autologous pericardium in such a manner as to fix the composite product to the site of the pericardial defect located between the epicardium and the sternum. The results of the evaluation are shown in table 3.

As is apparent from the above results, it was confirmed that the fibrosis inhibition and adhesion barrier kit as a product of the present invention can promote the self-organization of decellularized tissue to reconstitute autologous pericardial-like tissue having appropriate strength.

INDUSTRIAL APPLICABILITY

The present invention is useful as an inhibitor of fibrosis on the surface of organs or tissues, and also useful for inhibiting tissue adhesion.

Claims

1. An inhibitor of tissue fibrosis comprising a biocompatible polymer, wherein the biocompatible polymer is fibrin, wherein the biocompatible polymer is immobilized to a tissue to be inhibited from tissue fibrosis.

2. A tissue fibrosis inhibition kit comprising a combination of a biocompatible polymer and a cytoskeletal material, wherein the biocompatible polymer is fixed to one tissue to be inhibited from tissue fibrosis, and a tissue defect site in the other tissue is repaired with the cytoskeletal material.

3. The fibrosis inhibition kit according to claim 2, wherein said biocompatible polymer is fibrin or dextrin gel.

4. The fibrosis inhibition kit according to claim 2, wherein said cell scaffold material is decellularized tissue.

5. The fibrosis inhibition kit according to claim 4, wherein the decellularized tissue is a tissue made by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura, peritoneum, omentum majus, diaphragm, fascia, dermis, and skin.

6. The fibrosis inhibition kit according to claim 2, which promotes self-organization of the cell scaffold material.

7. The fibrosis suppression kit according to claim 2, wherein the tissue to be suppressed in tissue fibrosis is epicardium.

8. A tissue fibrosis inhibition kit comprising a combination of a biocompatible polymer and a pericardial substitute, wherein the biocompatible polymer is fixed to one tissue to be inhibited from tissue fibrosis, and a tissue defect site in the other tissue is repaired with the pericardial substitute.

9. The fibrosis inhibition kit according to claim 8, wherein said biocompatible polymer is fibrin or dextrin gel.

10. The fibrosis suppression kit of claim 8, wherein said pericardial substitute is decellularized tissue.

11. The fibrosis suppression kit according to claim 10, wherein the decellularized tissue is a tissue made by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura, peritoneum, omentum majus, diaphragm, fascia, dermis, and skin.

12. The fibrosis suppression kit of claim 8, which promotes self-organization of the pericardial substitute.

13. The fibrosis suppression kit according to claim 8, wherein the tissue to be suppressed in tissue fibrosis is pericardium.

14. An adhesion barrier kit in which a biocompatible polymer is fixed to one tissue to be inhibited from tissue adhesion, and a tissue defect site in the other tissue is repaired with a cell scaffold material or a pericardial substitute.

15. The adhesion barrier kit of claim 14, wherein the biocompatible polymer is fibrin or dextrin gel.

16. The adhesion barrier kit of claim 14, wherein the cell scaffold material or the pericardial substitute is decellularized tissue.

17. The adhesion barrier kit of claim 16, wherein the decellularized tissue is a tissue made by decellularizing a biological tissue selected from the group consisting of small intestine submucosa, pericardium, bladder, amnion, dura, peritoneum, omentum majus, diaphragm, fascia, dermis, and skin.

18. The adhesion barrier kit of any one of claims 14-17, which facilitates self-organization of the cell scaffold material or the pericardial substitute.

19. The adhesion barrier kit according to any one of claims 14-17, wherein the tissue to be inhibited from adhesion is epicardium.